scholarly journals Rhodobacter sphaeroides CarD Negatively Regulates Its Own Promoter

2021 ◽  
Author(s):  
Kemardo K. Henry ◽  
Wilma Ross ◽  
Richard L. Gourse
Keyword(s):  
Transcription Factor ◽  
Rna Polymerase ◽  
Rhodobacter Sphaeroides ◽  
Bioinformatic Analysis ◽  
Negative Regulation ◽  
Full Length ◽  
Kinetic Properties ◽  
Basal Activity ◽  
Promoter Escape

Bioinformatic analysis showed previously that a majority of promoters in the photoheterotrophic α-proteobacterium Rhodobacter sphaeroides lack the thymine at the last position of the -10 element (-7T), a base that is very highly conserved in promoters in bacteria other than α-proteobacteria. The absence of -7T was correlated with low promoter activity using purified R sphaeroides RNA polymerase (RNAP), but the transcription factor CarD compensated by activating almost all promoters lacking -7T tested in vitro , including rRNA promoters. Here we show that a previously uncharacterized R. sphaeroides promoter, the promoter for carD itself, has high basal activity relative to other tested R. sphaeroides promoters despite lacking -7T, and its activity is inhibited rather than activated by CarD. This high basal activity is dependent on a consensus extended -10 element (TGn) and specific features in the spacer immediately upstream of the extended -10 element. CarD negatively autoregulates its own promoter by producing abortive transcripts, limiting promoter escape and reducing full length mRNA synthesis. This mechanism of negative regulation differs from that employed by classical repressors, in which the transcription factor competes with RNA polymerase for binding to the promoter, and with the mechanism of negative regulation used by transcription factors like DksA/ppGpp and TraR that allosterically inhibit the rate of open complex formation. IMPORTANCE R. sphaeroides CarD activates many promoters by binding directly to RNAP and to DNA just upstream of the -10 element. In contrast, we show here that CarD inhibits its own promoter using the same interactions with RNAP and DNA used for activation. Inhibition results from increasing abortive transcript formation, thereby decreasing promoter escape and full-length RNA synthesis. We propose that the combined interactions of RNAP with CarD, with the extended -10 element, and with features in the adjacent -10/-35 spacer DNA stabilize the promoter complex, reducing promoter clearance. These findings support previous predictions that the effects of CarD on transcription can be either positive or negative, depending on the kinetic properties of the specific promoter.

The general transcription factor RAP30 binds to RNA polymerase II and prevents it from binding nonspecifically to DNA

1992 ◽  
Vol 12 (1) ◽  
pp. 30-37
Author(s):  
M T Killeen ◽  
J F Greenblatt
Keyword(s):  
Transcription Factor ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Small Subunit ◽  
Glutathione S Transferase ◽  
General Transcription Factor ◽  
Indirect Consequence ◽  
Transcription In Vitro ◽  
Sigma 70

RAP30/74 is a human general transcription factor that binds to RNA polymerase II and is required for initiation of transcription in vitro regardless of whether the promoter has a recognizable TATA box (Z. F. Burton, M. Killeen, M. Sopta, L. G. Ortolan, and J. F. Greenblatt, Mol. Cell. Biol. 8:1602-1613, 1988). Part of the amino acid sequence of RAP30, the small subunit of RAP30/74, has limited homology with part of Escherichia coli sigma 70 (M. Sopta, Z. F. Burton, and J. Greenblatt, Nature (London) 341:410-414, 1989). To determine which sigmalike activities of RAP30/74 could be attributed to RAP30, we purified human RAP30 and a RAP30-glutathione-S-transferase fusion protein that had been produced in E. coli. Bacterially produced RAP30 bound to RNA polymerase II in the absence of RAP74. Both partially purified natural RAP30/74 and recombinant RAP30 prevented RNA polymerase II from binding nonspecifically to DNA. In addition, nonspecific transcription by RNA polymerase II was greatly inhibited by RAP30-glutathione-S-transferase. DNA-bound RNA polymerase II could be removed from DNA by partially purified RAP30/74 but not by bacterially expressed RAP30. Thus, the ability of RAP30/74 to recruit RNA polymerase II to a promoter-bound preinitiation complex may be an indirect consequence of its ability to suppress nonspecific binding of RNA polymerase II to DNA.

The human RNA polymerase I structure reveals an HMG-like transcription factor docking domain specific to metazoans

2021 ◽  
Author(s):  
Julia L Daiß ◽  
Michael Pilsl ◽  
Kristina Straub ◽  
Andrea Bleckmann ◽  
Mona Höcherl ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Rna Polymerase ◽  
In Vitro Transcription ◽  
Cellular Growth ◽  
Hmg Box ◽  
Transcription Bubble ◽  
Transcription System ◽  
Pol I ◽  
Additional Domain

Transcription of the ribosomal RNA precursor by RNA polymerase (Pol) I is a major determinant of cellular growth and dysregulation is observed in many cancer types. Here, we present the purification of human Pol I from cells carrying a genomic GFP-fusion on the largest subunit allowing the structural and functional analysis of the enzyme across species. In contrast to yeast, human Pol I carries a single-subunit stalk and in vitro transcription indicates a reduced proofreading activity. Determination of the human Pol I cryo-EM reconstruction in a close-to-native state rationalizes the effects of disease-associated mutations and uncovers an additional domain that is built into the sequence of Pol I subunit RPA1. This "dock II" domain resembles a truncated HMG-box incapable of DNA-binding which may serve as a downstream-transcription factor binding platform in metazoans. Biochemical analysis and ChIP data indicate that Topoisomerase 2a can be recruited to Pol I via the domain and cooperates with the HMG-box domain containing factor UBF. These adaptations of the metazoan Pol I transcription system may allow efficient release of positive DNA supercoils accumulating downstream of the transcription bubble.

scholarly journals The Human Isoform of RNA Polymerase II Subunit hRPB11bα Specifically Interacts with Transcription Factor ATF4

2019 ◽  
Vol 21 (1) ◽  
pp. 135 ◽  
Author(s):  
Sergey A. Proshkin ◽  
Elena K. Shematorova ◽  
George V. Shpakovski
Keyword(s):  
Transcription Factor ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Fetal Brain ◽  
Gene Promoters ◽  
Α Subunit ◽  
Promoter Recognition ◽  
Rnap Ii ◽  
Human Specific

Rpb11 subunit of RNA polymerase II of Eukaryotes is related to N-terminal domain of eubacterial α subunit and forms a complex with Rpb3 subunit analogous to prokaryotic α2 homodimer, which is involved in RNA polymerase assembly and promoter recognition. In humans, a POLR2J gene family has been identified that potentially encodes several hRPB11 proteins differing mainly in their short C-terminal regions. The functions of the different human specific isoforms are still mainly unknown. To further characterize the minor human specific isoform of RNA polymerase II subunit hRPB11bα, the only one from hRPB11 (POLR2J) homologues that can replace its yeast counterpart in vivo, we used it as bait in a yeast two-hybrid screening of a human fetal brain cDNA library. By this analysis and subsequent co-purification assay in vitro, we identified transcription factor ATF4 as a prominent partner of the minor RNA polymerase II (RNAP II) subunit hRPB11bα. We demonstrated that the hRPB11bα interacts with leucine b-Zip domain located on the C-terminal part of ATF4. Overexpression of ATF4 activated the reporter more than 10-fold whereas co-transfection of hRPB11bα resulted in a 2.5-fold enhancement of ATF4 activation. Our data indicate that the mode of interaction of human RNAP II main (containing major for of hRPB11 subunit) and minor (containing hRPB11bα isoform of POLR2J subunit) transcription enzymes with ATF4 is certainly different in the two complexes involving hRPB3–ATF4 (not hRPB11a–ATF4) and hRpb11bα–ATF4 platforms in the first and the second case, respectively. The interaction of hRPB11bα and ATF4 appears to be necessary for the activation of RNA polymerase II containing the minor isoform of the hRPB11 subunit (POLR2J) on gene promoters regulated by this transcription factor. ATF4 activates transcription by directly contacting RNA polymerase II in the region of the heterodimer of α-like subunits (Rpb3–Rpb11) without involving a Mediator, which provides fast and highly effective activation of transcription of the desired genes. In RNA polymerase II of Homo sapiens that contains plural isoforms of the subunit hRPB11 (POLR2J), the strength of the hRPB11–ATF4 interaction appeared to be isoform-specific, providing the first functional distinction between the previously discovered human forms of the Rpb11 subunit.

scholarly journals A Region within the RAP74 Subunit of Human Transcription Factor IIF Is Critical for Initiation but Dispensable for Complex Assembly

1999 ◽  
Vol 19 (11) ◽  
pp. 7377-7387 ◽  
Author(s):  
Delin Ren ◽  
Lei Lei ◽  
Zachary F. Burton
Keyword(s):  
Transcription Factor ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Residual Activity ◽  
Dna Supercoiling ◽  
Single Amino Acid ◽  
Phosphodiester Bond ◽  
Promoter Escape ◽  
Transcription Factor Iif ◽  
Human Transcription Factor

ABSTRACT Human transcription factor IIF (TFIIF) is an α2β2 heterotetramer of RNA polymerase II-associating 74 (RAP74) and RAP30 subunits. Mutagenic analysis shows that the N-terminal region of RAP74 between L155 (leucine at codon 155) and M177 is important for initiation. Mutants in this region have reduced activity in transcription, but none are inactive. Single amino acid substitutions at hydrophobic residues L155, W164, I176, and M177 have similar activity to RAP74(1–158), from which all but three amino acids of this region are deleted. Residual activity can be explained because each of these mutants forms a complex with RAP30 and recruits RNA polymerase II into the preinitiation complex. Mutants are defective for formation of the first phosphodiester bond from the adenovirus major late promoter but do not appear to have an additional significant defect in promoter escape. Negative DNA supercoiling partially compensates for the defects of TFIIF mutants in initiation, indicating that TFIIF may help to untwist the DNA helix for initiation.

scholarly journals A Fully Recombinant System for Activator-dependent Archaeal Transcription

2004 ◽  
Vol 279 (50) ◽  
pp. 51719-51721 ◽  
Author(s):  
Mohamed Ouhammouch ◽  
Finn Werner ◽  
Robert O. J. Weinzierl ◽  
E. Peter Geiduschek
Keyword(s):  
Transcription Factor ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcriptional Regulators ◽  
The Core ◽  
General Transcription Factor ◽  
Core Components ◽  
Archaeal Transcription ◽  
Bacterial Type

The core components of the archaeal transcription apparatus closely resemble those of eukaryotic RNA polymerase II, while the DNA-binding transcriptional regulators are predominantly of bacterial type. Here we report the construction of an entirely recombinant system for positively regulated archaeal transcription. By omitting individual subunits, or sets of subunits, from thein vitroassembly of the 12-subunit RNA polymerase from the hyperthermophileMethanocaldococcus jannaschii, we describe a functional dissection of this RNA polymerase II-like enzyme, and its interactions with the general transcription factor TFE, as well as with the transcriptional activator Ptr2.

scholarly journals Heterogeneous nuclear ribonucleoprotein K is a transcription factor.

1996 ◽  
Vol 16 (5) ◽  
pp. 2350-2360 ◽  
Author(s):  
E F Michelotti ◽  
G A Michelotti ◽  
A I Aronsohn ◽  
D Levens
Keyword(s):  
Transcription Factor ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Hnrnp K ◽  
Heterogeneous Nuclear Ribonucleoprotein ◽  
Transcription In Vitro ◽  
Nuclear Ribonucleoprotein ◽  
Rna Polymerase Ii Transcription

The CT element is a positively acting homopyrimidine tract upstream of the c-myc gene to which the well-characterized transcription factor Spl and heterogeneous nuclear ribonucleoprotein (hnRNP) K, a less well-characterized protein associated with hnRNP complexes, have previously been shown to bind. The present work demonstrates that both of these molecules contribute to CT element-activated transcription in vitro. The pyrimidine-rich strand of the CT element both bound to hnRNP K and competitively inhibited transcription in vitro, suggesting a role for hnRNP K in activating transcription through this single-stranded sequence. Direct addition of recombinant hnRNP K to reaction mixtures programmed with templates bearing single-stranded CT elements increased specific RNA synthesis. If hnRNP K is a transcription factor, then interactions with the RNA polymerase II transcription apparatus are predicted. Affinity columns charged with recombinant hnRNP K specifically bind a component(s) necessary for transcription activation. The depleted factors were biochemically complemented by a crude TFIID phosphocellulose fraction, indicating that hnRNP K might interact with the TATA-binding protein (TBP)-TBP-associated factor complex. Coimmunoprecipitation of a complex formed in vivo between hnRNP K and epitope-tagged TBP as well as binding in vitro between recombinant proteins demonstrated a protein-protein interaction between TBP and hnRNP K. Furthermore, when the two proteins were overexpressed in vivo, transcription from a CT element-dependent reporter was synergistically activated. These data indicate that hnRNP K binds to a specific cis element, interacts with the RNA polymerase II transcription machinery, and stimulates transcription and thus has all of the properties of a transcription factor.

scholarly journals Interaction of the Vaccinia Virus RNA Polymerase-Associated 94-Kilodalton Protein with the Early Transcription Factor

2009 ◽  
Vol 83 (23) ◽  
pp. 12018-12026 ◽  
Author(s):  
Zhilong Yang ◽  
Bernard Moss
Keyword(s):  
Transcription Factor ◽  
Rna Polymerase ◽  
Vaccinia Virus ◽  
Transcription Initiation ◽  
Elongation Factor ◽  
Small Subunit ◽  
Functional Link ◽  
Early Transcription

ABSTRACT A multisubunit RNA polymerase (RPO) encoded by vaccinia virus (VACV), in conjunction with specific factors, transcribes early, intermediate, and late viral genes. However, an additional virus-encoded polypeptide referred to as the RPO-associated protein of 94 kDa (RAP94) is tightly bound to the RPO for the transcription of early genes. Unlike the eight RPO core subunits, RAP94 is synthesized exclusively at late times after infection. Furthermore, RAP94 is necessary for the packaging of RPO and other components needed for early transcription in assembling virus particles. The direct association of RAP94 with NPH I, a DNA-dependent ATPase required for transcription termination, and the multifunctional poly(A) polymerase small subunit/2′-O-methyltransferase/elongation factor was previously demonstrated. That RAP94 provides a structural and functional link between the core RPO and the VACV early transcription factor (VETF) has been suspected but not previously demonstrated. Using VACV recombinants that constitutively or inducibly express VETF subunits and RAP94 with affinity tags, we showed that (i) VETF associates only with RPO containing RAP94 in vivo and in vitro, (ii) the association of RAP94 with VETF requires both subunits of the latter, (iii) neither viral DNA nor other virus-encoded late proteins are required for the interaction of RAP94 with VETF and core RPO subunits, (iv) different domains of RAP94 bind VETF and core subunits of RPO, and (v) NPH I and VETF bind independently and possibly simultaneously to the N-terminal region of RAP94. Thus, RAP94 provides the bridge between the RPO and proteins needed for transcription initiation, elongation, and termination.

scholarly journals Abstract P-18: Phosphorylation of RNA Polymerase II C-Terminal Domain Affects Transcript Elongation Through Chromatin

2021 ◽  
Vol 11 (Suppl_1) ◽  
pp. S19-S19
Author(s):  
Sohail Akhtar ◽  
Elena Kotova ◽  
Nadezhda Gerasimova ◽  
Vasily Studitsky
Keyword(s):  
Rna Polymerase ◽  
Chromatin Structure ◽  
Full Length ◽  
Pol Ii ◽  
Terminal Domain ◽  
Significant Difference ◽  
Transcript Elongation ◽  
Hyperphosphorylated Form ◽  
Ctd Phosphorylation

Background: Transcription is the central point of gene regulation where the efficient maintenance of chromatin structure during the passage of RNA polymerase (Pol II) is critical for cell survival and functioning. The phosphorylation of carboxy-terminal domain (CTD) of the large subunit (Rpb1) of Pol II plays a key role in transcription through chromatin providing the binding and dissociation of factors essential for the mRNA biogenesis. Although the regulatory effect of chromatin structure on multiple stages of transcription has been well established, the role of CTD phosphorylation itself has not been systematically addressed. Methods: The effect of differentially phosphorylated Pol II-CTD on transcript elongation through chromatin was studied using in vitro transcription system based on mononucleosomes precisely positioned on DNA. The unphosphorylated and hyperphosphorylated Pol II-CTD were obtained using yeast genetics as well as in vitro kinase or phosphatases. Transcription rate and positions of pausing were measured using authentic elongation complexes comprising Pol II having different CTD phosphorylation states. The quantitative analysis of the transcripts was conducted using denaturing PAGE. Results: We observed a significant difference in the transcription through chromatin depending on CTD phosphorylation level. Thus, experiments on transcription of nucleosomes with Pol II isoforms have shown that the hyperphosphorylated form more efficiently transcribes the nucleosome and leads to a faster accumulation of the full-length RNA product than the non-phosphorylated isoform of Pol II. The non-phosphorylated isoform of the enzyme is characterized by a stronger pause in the early nucleosomal region and a slower accumulation of the full-length RNA product. Conclusion: Hyperphosphorylated form more efficiently transcribes the nucleosome and leads to a faster accumulation of the full-length RNA product as compared with the non-phosphorylated isoform of Pol II. A preliminary model of the effect of Pol II hyperphosphorylation on nucleosomal DNA transcription is proposed.

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